In recent years, spectroscopic analysis has come to be widely used as a method for carrying out analysis or measurement on semiconductors, biological samples, various types of liquid sample, and so on. However, with a conventional spectroscopic analysis method, in the case of analyzing a very small amount of a substance or a very small sample in a very small space, it has been necessary to carry out the measurement in a vacuum environment. Moreover, there has been a problem that the sample may be damaged or destroyed upon using an electron beam or ion beam.
Moreover, when handling an extremely small amount of a sample in a solution, biological tissue, or the like, it is essential to use an optical microscope that enables analysis to be carried out with high precision and high spatial resolution. The only type of such microscope actually used is a laser fluorescence microscope. The target of analysis is thus naturally limited to being a molecule that is fluorescent with a laser fluorescence microscope.
Due to this state of affairs, there have been demands for an analysis method according to which a vacuum environment is not required, analysis can be carried out without contacting or risking damaging the sample, and moreover the target of analysis is not limited to being a fluorescent molecule, and analysis can be carried out with high precision and high spatial resolution.
A photothermal conversion spectroscopic analysis method that uses a thermal lens effect brought about by photothermal conversion is attracting attention as an analysis method that satisfies these demands.
This photothermal conversion spectroscopic analysis method uses a photothermal conversion effect in which light is convergently irradiated onto a sample, whereupon a solute in the sample absorbs the light, and hence the temperature of the solvent is locally raised by thermal energy released due to the absorbed light, whereby the refractive index changes, and hence a thermal lens is formed.
FIG. 3 is a view useful in explaining the principle of a thermal lens.
In FIG. 3, exciting light is convergently irradiated onto an extremely small sample via an objective lens of a microscope, whereby a photothermal conversion effect is brought about. For most substances, the refractive index drops as the temperature rises. Consequently, in the sample onto which the exciting light has been convergently irradiated, the refractive index drops, with the drop in the refractive index being larger closer to the center of the converged light, which is where the extent of the rise in temperature is largest; moving away from the center of the converged light toward the periphery, the extent of the rise in temperature becomes smaller due to thermal diffusion, and hence the drop in refractive index becomes smaller. Optically, the resulting refractive index distribution produces exactly the same effect as a concave lens, and hence the effect is referred to as the thermal lens effect. The size of the thermal lens effect, i.e. the power of the concave lens, is proportional to the optical absorbance of the sample. Moreover, in the case that the refractive index increases with temperature, a similar effect is produced, but conversely the thermal lens is convex.
In the photothermal conversion spectroscopic analysis method described above, thermal diffusion in the sample, i.e. change in the refractive index of the sample, is observed, and hence the method is suitable for detecting concentrations in extremely small samples.
A photothermal conversion spectroscopic analysis apparatus that carries out the photothermal conversion spectroscopic analysis method described above is described, for example, in Japanese Laid-open Patent Publication (Kokai) No. 10-232210.
In such a photothermal conversion spectroscopic analysis apparatus, the sample is disposed below the objective lens of a microscope, and exciting light of a predetermined wavelength outputted from an exciting light source is introduced into the microscope, and thus convergently irradiated via the objective lens onto an extremely small region in the sample. A thermal lens is thus formed centered on the focal position of the convergently irradiated exciting light.
Moreover, detecting light having a wavelength different to that of the exciting light is emitted from a detecting light source, and is introduced into the microscope, before exiting from the microscope. The detecting light that has exited from the microscope is convergently irradiated onto the thermal lens that has been formed in the sample by the exciting light. Upon passing through the sample, the detecting light is diverged or converged by the effect of the thermal lens. The diverged or converged detecting light exiting from the sample is taken as signal light, and passes through a converging lens and a filter, or just a filter, before being received by a detector and thus detected. The intensity of the detected signal light depends on the refractive index of the thermal lens formed in the sample.
The frequency of the detecting light may be the same as that of the exciting light, or the exciting light may also be used as the detecting light. In general, good sensitivity is obtained in the case that the exciting light and the detecting light are made to have different frequencies to one another.
However, with a photothermal conversion spectroscopic analysis apparatus as described above, the structure of the optical system and so on for the light sources, the measurement section and the detection section (photoelectric conversion section) is complex, and hence such an apparatus has been large in size and has thus lacked portability. Consequently, there is a problem in that when carrying out analysis or a chemical reaction using such a photothermal conversion spectroscopic analysis apparatus, there are limitations with regard to the installation site of the apparatus and the operation of the apparatus, and hence there is a problem of the work efficiency for a user being poor.
In many cases of using a photothermal conversion spectroscopic analysis method that makes use of a thermal lens, it is necessary for the focal position of the exciting light and the focal position of the detecting light to be different to one another. FIGS. 4A and 4B are views useful in explaining the formation position of a thermal lens and the focal position of detecting light in the direction of travel of exciting light; FIG. 4A shows a case in which an objective lens has chromatic aberration, and FIG. 4B shows a case in which the objective lens does not have chromatic aberration.
In the case that the objective lens 130 has chromatic aberration, as shown in FIG. 4A, the thermal lens 131 is formed at the focal position 132 of the exciting light, and the focal position 133 of the detecting light is shifted by an amount ΔL from the focal position 132 of the exciting light; changes in the refractive index of the thermal lens 131 can thus be detected as changes in the focal distance of the detecting light. On the other hand, in the case that the objective lens 130 does not have chromatic aberration, as shown in FIG. 4B, the focal position 133 of the detecting light is almost exactly the same as the focal position 132 of the exciting light, i.e. the position of the thermal lens 131. As a result, the detecting light is not refracted by the thermal lens 131, and hence changes in the refractive index of the thermal lens 131 cannot be detected.
However, the objective lens of a microscope is generally manufactured so as not to have chromatic aberration, and hence for the reason described above, the focal position 133 of the detecting light is almost exactly the same as the position of the thermal lens 131 formed at the focal position 132 of the exciting light (FIG. 4B). Changes in the refractive index of the thermal lens 131 thus cannot be detected. There is thus a problem in that the position of the sample where the thermal lens is formed must be shifted from the focal position 133 of the detecting light every time measurement is carried out as shown in FIG. 5A or 5B, to, for example, shift a focal position of the detecting light to a position 134 as shown in FIG. 5A. Alternatively, the detecting light must be diverged or converged slightly using a lens (not shown) before being introduced into the objective lens 130 so that the focal position 133 of the detecting light is shifted from the thermal lens 131 as shown in FIG. 6. This requires time and effort, and hence the work efficiency for a user is poor.
It is an object of the present invention to provide a photothermal conversion spectroscopic analysis method that enables measurement to be carried out with high sensitivity, and a small-sized photothermal conversion spectroscopic analysis apparatus that carries out the method.